Reactivation of TWIST1 contributes to Ewing sarcoma metastasis.
ABSTRACT: BACKGROUND:Ewing sarcoma is a cancer of bone and soft tissue. Despite aggressive treatment, survival remains poor, particularly in patients with metastatic disease. Failure to treat Ewing sarcoma is due to the lack of understanding of the molecular pathways that regulate metastasis. In addition, no molecular prognostic markers have been identified for Ewing sarcoma to risk stratify patients. PROCEDURE:Ewing sarcoma patients were divided into high or low Twist1 gene expression and survival curves were generated using the R2 microarray-based Genomic Analysis platform (http://r2.amc.nl). Tumors from Ewing sarcoma patients were also evaluated for TWIST1 expression by immunohistochemistry. Ewing sarcoma xenografts were established to evaluate the role of TWIST1 in metastasis. The effects of Twist1 on migration and invasion were evaluated using migration and invasion assays in A673 and RDES cells. RESULTS:Twist1 expression was a negative prognostic marker for overall survival in a public Ewing sarcoma patient data set based on Twist1 mRNA levels and in patient tumor samples based on Twist1 immunohistochemistry. TWIST1 is detected in significantly higher percentage of patients with metastatic diseases than localized disease. Using Ewing sarcoma tumor xenografts in mice, we found that suppressing TWIST1 levels suppressed metastasis without affecting primary tumor development. Knockdown of Twist1 inhibited the migration and invasion capability, while overexpression of Twist1 promoted migration and invasion in Ewing sarcoma cells. CONCLUSION:These results suggest that TWIST1 promotes metastasis in Ewing sarcoma and could be used as a prognostic marker for treatment stratification; however, further validation is required in a larger cohort of patients.
Project description:Ewing Sarcoma is the second most common solid pediatric malignant neoplasm of bone and soft tissue. Driven by EWS/Ets, or rarely variant, oncogenic fusions, Ewing Sarcoma is a biologically and clinically aggressive disease with a high propensity for metastasis. However, the mechanisms underpinning Ewing Sarcoma metastasis are currently not well understood. In the present study, we identify and characterize a novel metastasis-promotional pathway in Ewing Sarcoma, involving the histone demethylase KDM3A, previously identified by our laboratory as a new cancer-promoting gene in this disease. Using global gene expression profiling, we show that KDM3A positively regulates genes and pathways implicated in cell migration and metastasis, and demonstrate, using functional assays, that KDM3A promotes migration in vitro and experimental, post-intravasation, metastasis in vivo. We further identify the melanoma cell adhesion molecule (MCAM) as a novel KDM3A target gene in Ewing Sarcoma, and an important effector of KDM3A pro-metastatic action. Specifically, we demonstrate that MCAM depletion, like KDM3A depletion, inhibits cell migration in vitro and experimental metastasis in vivo, and that MCAM partially rescues impaired migration due to KDM3A knock-down. Mechanistically, we show that KDM3A regulates MCAM expression both through a direct mechanism, involving modulation of H3K9 methylation at the MCAM promoter, and an indirect mechanism, via the Ets1 transcription factor. Finally, we identify an association between high MCAM levels in patient tumors and poor survival, in two different Ewing Sarcoma clinical cohorts. Taken together, our studies uncover a new metastasis-promoting pathway in Ewing Sarcoma, with therapeutically targetable components.
Project description:Metastatic Ewing sarcoma has a very poor prognosis and therefore new investigations into the biologic drivers of metastatic progression are key to finding new therapeutic approaches. The tumor microenvironment is highly dynamic, leading to exposure of different regions of a growing solid tumor to changes in oxygen and nutrient availability. Tumor cells must adapt to such stress in order to survive and propagate. In the current study, we investigate how Ewing sarcoma cells respond to the stress of growth factor deprivation and hypoxia. Our findings reveal that serum deprivation leads to a reversible change in Ewing cell cytoskeletal phenotypes. Using an array of migration and invasion techniques, including gelatin matrix degradation invadopodia assays, we show that exposure of Ewing sarcoma cells to serum deprivation and hypoxia triggers enhanced migration, invadopodia formation, matrix degradation and invasion. Further, these functional changes are accompanied by and dependent on activation of Src kinase. Activation of Src, and the associated invasive cell phenotype, were blocked by exposing hypoxia and serum-deprived cells to the Src inhibitor dasatinib. These results indicate that Ewing sarcoma cells demonstrate significant plasticity in response to rapidly changing micro-environmental stresses that can result from rapid tumor growth and from necrosis-causing therapies. In response to these stresses, Ewing cells transition to a more migratory and invasive state and our data show that Src is an important mediator of this stress response. Our data support exploration of clinically available Src inhibitors as adjuvant agents for metastasis prevention in Ewing sarcoma.
Project description:Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma.
Project description:Ewing sarcoma is an aggressive malignant neoplasm with high propensity for metastasis and poor clinical outcomes. The EWS/Fli1 oncofusion is the disease driver in > 90% of cases, but presents a difficult therapeutic target. Moreover, EWS/Fli1 plays a complex role in disease progression, with inhibitory effects on critical steps of metastasis. Like many other pediatric cancers, Ewing sarcoma is a disease marked by epigenetic dysregulation. Epigenetic mechanisms present alternative targeting opportunities, but their contributions to Ewing sarcoma metastasis and disease progression remain poorly understood. Here, we show that the epigenetic regulators KDM5A and PHF2 promote growth and metastatic properties in Ewing sarcoma, and, strikingly, activate expression many pro-metastatic genes repressed by EWS/Fli1. These genes include L1CAM, which is associated with adverse outcomes in Ewing sarcoma, and promotes migratory and invasive properties. KDM5A and PHF2 retain their growth promoting effects in more metastatically potent EWS/Fli1low cells, and PHF2 promotes both invasion and L1CAM expression in this cell population. Furthermore, KDM5A and PHF2 each contribute to the increased metastatic potency of EWS/Fli1low cells in vivo. Together, these studies identify KDM5A and PHF2 as novel disease-promoting factors, and potential new targets, in Ewing sarcoma, including the more metastatically potent EWS/Fli1low cell population.
Project description:Cancer-associated fibroblasts (CAFs) play important roles in cancer progression. Twist1 was recently reported to be a key regulator of CAFs in gastric cancer, but its role in other types of cancer remains unclear, especially for esophageal squamous cell carcinoma (ESCC). We assessed the Twist1 expression on stromal fibroblasts using immunohistochemistry in 169 tissue specimens from ESCC patients, and performed in vitro and in vivo experiments to confirm the role of Twist1 in CAFs of ESCC. And we investigated the biological pathways that are activated in Twist1-high ESCC using The Cancer Genome Atlas (TCGA) data. The expression of Twist1 in stromal fibroblasts was observed in 89.9% of ESCC patients and positively associated with the increased depth of tumor invasion, lymph node metastasis, and advanced clinical stage, and a significant adverse prognostic factor in overall survival. Twist1-expressing stromal fibroblasts also expressed representative CAF markers, and co-localization of Twist1 and CAF markers were confirmed by confocal immunofluorescence imaging. Bioinformatic analysis of mRNA expression data of esophageal cancer from TCGA revealed that gene sets of CAFs were highly enriched in Twist1-high ESCC. Depletion of Twist1 in ex vivo cultured ESCC CAFs induced significant decrease in migration, invasion, colony formation, sphere formation, and contractibility of ESCC cancer cells compared to control CAFs. Furthermore, Twist1-expressing fibroblasts remarkably enhanced the in vivo tumorigenicity of ESCC in a xenograft model. In conclusion, Twist1 could be a novel CAF marker for the prognostic evaluation of ESCC patients as well as a potent therapeutic target for ESCC.
Project description:Ewing sarcoma (ES) is the second most common bone malignancy affecting children and young adults with poor prognosis due to high metastasis incidence. Our group previously described that EphA2, a tyrosine kinase receptor, promotes angiogenesis in Ewing sarcoma (ES) cells via ligand-dependent signaling. Now we wanted to explore EphA2 ligand-independent activity, controlled upon phosphorylation at S897 (p-EphA2S897 ), as it has been linked to metastasis in several malignancies. By reverse genetic engineering we explored the phenotypic changes after EphA2 removal or reintroduction. Gene expression microarray was used to identify key players in EphA2 signaling. Mice were employed to reproduce metastatic processes from orthotopically implanted engineered cells. We established a correlation between ES cells aggressiveness and p-EphA2S897 . Moreover, stable overexpression of EphA2 in low EphA2 expression ES cells enhanced proliferation and migration, but not a non-phosphorylable mutant (S987A). Consistently, silencing of EphA2 reduced tumorigenicity, migration and invasion in vitro, and lung metastasis incidence in experimental and spontaneous metastasis assays in vivo. A gene expression microarray revealed the implication of EphA2 in cell signaling, cellular movement and survival. ADAM19 knockdown by siRNA technology strongly reproduced the negative effects on cell migration observed after EphA2 silencing. Altogether, our results suggest that p-EphA2S897 correlates with aggressiveness in ES, so blocking its function may be a promising treatment.
Project description:The heterogeneous breast cancers can be classified into different subtypes according to their histopathological characteristics and molecular signatures. Foxa1 expression is linked with luminal breast cancer (LBC) with good prognosis, whereas Twist1 expression is associated with basal-like breast cancer (BLBC) with poor prognosis owing to its role in promoting epithelial-to-mesenchymal transition (EMT), invasiveness and metastasis. However, the regulatory and functional relationships between Twist1 and Foxa1 in breast cancer progression are unknown. In this study, we demonstrate that in the estrogen receptor (ER?)-positive LBC cells Twist1 silences Foxa1 expression, which has an essential role in relieving Foxa1-arrested migration, invasion and metastasis of breast cancer cells. Mechanistically, Twist1 binds to Foxa1 proximal promoter and recruits the NuRD transcriptional repressor complex to de-acetylate H3K9 and repress RNA polymerase II recruitment. Twist1 also silences Foxa1 promoter by inhibiting AP-1 recruitment. Twist1 expression in MCF7 cells silenced Foxa1 expression, which was concurrent with the induction of EMT, migration, invasion and metastasis of these cells. Importantly, restored Foxa1 expression in these cells largely inhibited Twist1-promoted migration, invasion and metastasis. Restored Foxa1 expression did not change the Twist1-induced mesenchymal cellular morphology and the expression of Twist1-regulated E-cadherin, ?-catenin, vimentin and Slug, but it partially rescued Twist1-silenced ER? and cytokeratin 8 expression and reduced Twist1-induced integrin ?5, integrin ?1 and MMP9 expression. In a xenografted mouse model, restored Foxa1 also increased Twist1-repressed LBC markers and decreased Twist1-induced BLBC markers. Furthermore, Twist1 expression is negatively correlated with Foxa1 in the human breast tumors. The tumors with high Twist1 and low Foxa1 expressions are associated with poor distant metastasis-free survival. These results demonstrate that Twist1's silencing effect on Foxa1 expression is largely responsible for Twist1-induced migration, invasion and metastasis, but less responsible for Twist1-induced mesenchymal morphogenesis and expression of certain EMT markers.
Project description:Proteins that promote epithelial to mesenchymal transition (EMT) are associated with cancer metastasis. Inhibition of EMT regulators may be a promising approach in cancer therapy. In this study, Thymoquinone (TQ) was used to treat cancer cell lines to investigate its effects on EMT-regulatory proteins and cancer metastasis. We show that TQ inhibited cancer cell growth, migration and invasion in a dose-dependent manner. At the molecular level, TQ treatment decreased the transcriptional activity of the TWIST1 promoter and the mRNA expression of TWIST1, an EMT-promoting transcription factor. Accordingly, TQ treatment also decreased the expression of TWIST1-upregulated genes such as N-Cadherin and increased the expression of TWIST1-repressed genes such as E-Cadherin, resulting in a reduction of cell migration and invasion. TQ treatment also inhibited the growth and metastasis of cancer cell-derived xenograft tumors in mice but partially attenuated the migration and invasion in TWIST1-overexpressed cell lines. Furthermore, we found that TQ treatment enhanced the promoter DNA methylation of the TWIST1 gene in BT 549 cells. Together, these results demonstrate that TQ treatment inhibits TWIST1 promoter activity and decreases its expression, leading to the inhibition of cancer cell migration, invasion and metastasis. These findings suggest TQ as a potential small molecular inhibitor of cancer growth and metastasis.
Project description:Ewing sarcoma is the second most common osseous disease in children and adolescents. It presents with a poor prognosis due to the high degree of malignancy and distant metastasis. In order to predict the disease prognosis and investigate a suitable therapeutic strategy for Ewing sarcoma, the present study aimed to describe the clinical characteristics, and to construct and validate nomograms for patients with non-metastatic Ewing sarcoma. A total of 627 cases of non-metastatic Ewing sarcoma were retrospectively collected from the Surveillance, Epidemiology, and End Results database between 2005 and 2014. Survival analysis and a machine learning model were used to identify independent prognostic variables and establish nomograms to estimate overall survival (OS) and cause-specific survival (CSS). The nomograms were bootstrap internally validated and externally validated using non-metastatic Ewing sarcoma cases from the First Affiliated Hospital of Zhengzhou University. The accuracy was also assessed by comparing with current American Joint Committee on Cancer (AJCC) staging systems. The total series consisted of 627 patients with non-metastatic Ewing sarcoma with a mean age of 20.14 years. Age, tumor extension, sex, International Classification of Diseases for Oncology, 3rd Edition histology, surgery and chemotherapy were identified as independent risk factors for OS and CSS. The aforementioned outcomes were incorporated to construct the nomograms, and the concordance indices (C-indices) for internal validation of OS and CSS prediction were 0.791 and 0.813, which were higher than those for AJCC sixth edition (OS, 0.531; CSS, 0.534) and seventh edition (OS, 0.547; CSS, 0.561), while the C-indices for external validation of OS and CSS prediction were 0.834 and 0.825, respectively. In conclusion, age, sex, tumor extension and surgery were independent prognostic factors for both OS and CSS. In addition, with regard to OS, the Ewing sarcoma subtype was a poor factor and chemotherapy was a favorable one. Nomograms based on reduced Cox models attained a satisfactory accuracy in predicting the survival of patients with non-metastatic Ewing sarcoma and could assist clinicians in evaluating survival more accurately.
Project description:Breast cancer is the leading cause of cancer-related deaths in the United States. The majority of deaths (90%) in breast cancer patients is caused by invasion and metastasis-two features related to the epithelial-to-mesenchymal transition (EMT). Twist1 is a key transcription factor that promotes the EMT, which leads to cell migration, invasion, cancer metastasis, and therapeutic resistance. Harmine is a beta-carboline alkaloid found in a variety of plants and was recently shown to be able to induce degradation of Twist Family BHLH Transcription Factor 1 (Twist1) in non-small cell lung cancer cells (NSCLC). In this study, we show that harmine can inhibit migration and invasion of both human and mouse breast cancer cells in a dose-dependent manner. Further study shows that this inhibition is most likely achieved by inducing a proteasome-dependent Twist1 degradation. At the concentrations tested, harmine did not affect the viability of cells significantly, suggesting that its inhibition of cancer cell migration and invasion is largely independent of its cytotoxicity, but due to its ability to affect regulators of EMT such as Twist1. This result may facilitate the development of strategies that target Twist1 to treat metastatic breast cancer, as Twist1 is expressed at a high level in metastatic breast cancer cells but not in normal cells.